The goals of this study are two-fold: 1) to understand how energy-dependent ATPase enzymes function in bacterial secretion and 2) to elucidate mechanisms of ATPase regulation by accessory proteins. This application will focus on characterizing the biochemical function of CIpV, a member of the Clp/Hsp100 family of AAA+ (ATPases associated with various cellular activities) protein unfoldases and remodeling enzymes. Clp/Hsp100 ATPases use the energy of ATP hydrolysis to unfold and translocate proteins through the pore of a hexameric assembly. Interestingly, protein secretion by the type VI secretion system (T6SS) in pathogenic proteobacteria requires CIpV and appears to be tightly controlled by a novel serine-threonine kinase pathway recently discovered in Pseudomonas aeruginosa. Despite a lack of biochemical evidence, current models of type VI secretion propose CIpV provides the motive force for protein translocation across the cell envelope. Furthermore, a phosphorylated accessory protein may regulate CIpV activity. To test these models, I will determine how general principles that govern characterized Clp/Hsp100 ATPases apply to CIpV biochemical function. Specifically, I will determine if CIpV is a highly specific protein unfoldase that binds and unfolds secreted T6SS proteins by measuring ATPase and protein unfolding activities. I will further characterize the sequence determinants that allow CIpV to recognize substrates. I will also determine if a phosphorylated accessory protein modulates CIpV enzyme activity and define the mechanism of regulation. I propose to solve the three-dimensional structure of this putative regulatory protein bound to elements of CIpV or CIpV substrates in order to understand the mechanism of regulation. PUBLIC HEALTH RELEVANCE: Protein secretion is an important process required for bacterial infection. This work proposes to biochemically characterize an enzyme important for protein secretion by several important pathogenic bacteria, particularly bacteria that infect patients with weakened immune systems. Knowledge gained from this study will further our understanding of the strategies employed by pathogenic bacteria for infection and will aid in the development of novel therapeutic strategies that specifically target protein secretion.